Simultaneous quantification of niacin and its three main metabolites in human plasma by LC–MS/MS

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2012-09, Vol.904, p.107-114
• Main Authors: Liu, Man, Zhang, Dan, Wang, Xiaolin, Zhang, Lina, Han, Jing, Yang, Man, Xiao, Xue, Zhang, Yanan, Liu, Huichen
• Format: Article
• Language: English
• Subjects: acetonitrile; Adult; Blanks; Calibration; centrifugation; Chromatography; Chromatography, Liquid - methods; Delayed-Action Preparations; Dosing; Drug Stability; Female; formic acid; Guidelines; Human; Humans; ionization; Linear Models; liquid chromatography; Male; Metabolites; Monitoring; N-methyl-2-pyridone-5-carboxamide; Niacin; Niacin - blood; Niacin - chemistry; Niacin - pharmacokinetics; Nicotinamide; Nicotinic Acids - blood; Nicotinic Acids - chemistry; Nicotinic Acids - pharmacokinetics; Nicotinuric acid; Pharmacokinetics; Pyridones - blood; Pyridones - chemistry; Pyridones - pharmacokinetics; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization; tandem mass spectrometry; Tandem Mass Spectrometry - methods
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2012.07.030

► A LC–MS/MS method for simultaneous quantification of four analytes was developed. ► Simple protein precipitation was employed for the sample preparation. ► The concentrations of endogenous NAM and 2-Pyr were determined. ► The rLLOQ of endogenous NAM and 2-Pyr was calculated. ► The interferences from isotope effect or endogenous substances were avoided. A sensitive and specific LC–MS/MS method for the simultaneous quantification of niacin (NA) and its three main metabolites nicotinamide (NAM), nicotinuric acid (NUA) and N-methyl-2-pyridone-5-carboxamide (2-Pyr) in human plasma has been developed and validated. Plasma samples (200μL) were prepared by deproteinization with acetonitrile (500μL), then the supernatant after centrifugation was evaporated and reconstituted. Chromatography was performed on a phenomenex synergi hydro-RP column with an isocratic elution of methanol-0.1% formic acid (5:95, v/v). The full separation of all analytes was achieved within 9min. Multiple-reaction monitoring (MRM) using the fragmentation transitions of m/z 124.1→80.1, 123.1→80.0, 181.0→79.0 and 153.1→110.2 in positive electrospray ionization (ESI) mode was performed to quantify NA, NAM, NUA and 2-Pyr, respectively. The calibration curves were linear over the concentration range of 2.0–3000ng/mL for NA and NUA, 10.0–1600ng/mL for NAM and 50.0–5000ng/mL for 2-Pyr. This method has been validated in accordance with the US FDA guidelines for bioanalytical method development and applied to the determination of NA and its three main metabolites in Chinese subjects following a single oral dose of niacin extended-release and simvastatin 1000mg/20mg. In particular, because of the endogenous NAM and 2-Pyr in human plasma, the concentrations of NAM and 2-Pyr in human plasma after dosing were determined by subtracting blank values of them.